Shiga-like toxins (SLTs) are produced by enterohemorrhagic Escherichia coli (EHEC), a group of organisms that cause food-borne hemorrhagic colitis and the hemolytic uremic syndrome. SLTs are comprised of pentamers of B subunits that bind to glycolipid receptors and monomers of A subunits that act as 28S rRNA N-glycosidases. In addition to expression of one or more types of SLTs, prototypic EHEC E. coli 0157:H7 strains also carry an ~90 kb plasmid as well as a gene, designated eae, that shares homology with the attach/efface eae gene of enteropathogenic E. coli (EPEC The long-term objective of this project is to define at the cellular and molecular levels the pathogenic mechanisms of SLT-producing E. coli. The SPECIFIC AIMS designed to achieve this goal are to: 1.) evaluate the basis for the marked virulence of EHEC 091:H21 strain B2F1 in the orally-infected, streptomycin-treated mouse model by examining the contribution to pathogenicity of slt-IIc operon copy number, the large plasmid, the intestinal site and level of colonization, and any adhesions identified by tnphoA mutagenesis; 2.) dissect the role of eae in EHEC adherence by constructing an eae negative derivative of an EHEC 0157:H7 strain and comparing the mutant with wild-type for adherence to HEp-2 cells and for virulence in the mouse model; 3.) analyze the basis for the differential Vero cell cytotoxicities of SLT-II and SLT-IIc by comparing the toxins for functional receptor, affinity of receptor binding, and influence of proteolytic cleavage on Vero cell cytotoxicity; 4.) define any environmental conditions that regulate expression of slt-II and slt- IIc in EHEC isolates by measuring chloramphenicol acetyltransferase (CAT) activity in promoter-cat fusions in vitro and in vivo; and 5.) investigate the reason(s) that E. coli strains that produce SLT-IIc or SLT-II alone are more virulent in the mouse model than strains that produce both SLT-I and SLT-II by assessing whether SLT-I interferes with the toxicity of SLT-IIc or SLT-II.